There currently exist a number of different probes that may be used for investigating cellular processes in vivo and in vitro. Most of these probes, and the techniques associated therewith fall into one of the following two categories:
a) radioactive, fluorescent or other readily-detectable labels that may be inserted or embedded into the cellular framework, such that specific chemical pathways and/or individual molecular entities may be studied;
b) simple visualization tools for investigating activity, either within cellular compartments or at the level of the whole cell.
There is, however, a need for techniques that can enable real-time reporting of biological or chemical events that may affect the function and/or morphology and/or structural properties of either the cell membrane, or alternatively, the function, morphology and viability of the whole cell. A key feature of these desired techniques is that they will not be limited to any specific molecule, molecular pathway or specific cell type or cell line.
A number of prior art publications disclose the use of polydiacetylene-based means for detecting membrane-perturbing events and/or agents:
WO 98/39632 suggests the use of polydiacetylenes for detecting reactions, by means of exposing the reaction means to a biopolymeric material comprising said polydiacetylenes. Preferably, the biopolymeric material is provided in the form of liposomes, films, tubules and other membrane-simulating forms.
WO 99/10743 describes the encapsulation of polydiacetylenes into metal oxide glass, and the use of the transparent composite obtained for the detection of various analytes.
WO 00/55623 discloses a beneficial combination of polydiacetylenes, lipids and suitable means linked thereto for detecting the presence of analytes in a liquid sample, wherein said analytes cannot react chemically with said polydiacetylenes and lipids. Specifically mentioned analytes include metal ions, biological ligands and peptides.
It is to be noted, however, that none of the foregoing publications disclose the use of the polydiacetylene construct in living cells.
Other publications have disclosed detection systems for use inside the outer membranes of living cells. WO 95/27204, for example, discloses a method for detecting and measuring molecules located at the cell surface (e.g. receptors), wherein said method involves labeling the cells with a molecular probe comprising a lipophilic moiety and a reporter moiety, preferably a fluorescence-emitting reporter moiety.
It is a purpose of the present invention to provide a method for “engineering” cells such that they contain within their outer membrane a membrane perturbation-detecting construct.
It is another purpose of the invention to provide the aforementioned engineered cells in a viable form such that they may be used as an assay system for detecting and/or measuring changes in the conformation and/or function of their cell membrane and processes occurring at the membrane surface.
It is a further purpose of the invention to provide a method for detecting and/or measuring the presence of agents and conditions that cause changes in cell membrane function and/or structure and processes occurring at the membrane surface, said method involving the use of the aforementioned engineered cells.
Further objects and advantages of the present invention will become apparent as the description proceeds.